Biofilm dysbiosis and caries activity: a surface or an individual issue?

Abstract Objective This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. Methodology A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Results Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Conclusion Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

Manejo de cárie radicular: um guia para o dentista brasileiro baseado na tradução e adaptação cultural do consenso internacional/ORCA E EFCD / Management of root caries: a guide for the Brazilian dentist based on the translation and cultural adaptation of the international consensus/ORCA and EFCD

Objetivo: A cárie radicular é um problema da Odontologia moderna, porém é notável a falta de diretrizes sobre o seu manejo. Objetivamos elaborar e adaptar um guia a partir da tradução das recomendações do consenso in-ternacional European Organization for Caries Research (ORCA) e European Federation of Conservative Dentistry (EFCD) para as tomadas de decisão clínica na intervenção do processo de cárie na pessoa idosa, com foco na cárie radicular. Materiais e métodos: O protocolo de tradução das recomendações do consenso internacional consistiu nas etapas: (1) tradução inicial, (2) síntese da tradução, (3) retradução, (4) revisão por comitê de especialistas, com adaptação cultural. A partir da tradução, foi desenvolvido um guia com diretrizes para tratamento de cárie radicular no Brasil. Resultados: Para prevenção de novas lesões é recomendada a escovação diária com dentifrício >1.500ppm/F. Dentifrícios com 5.000ppm/F ou vernizes (>20.000ppm/F) podem ser indicados para paralisar lesões radiculares ativas e para prevenção em pessoas idosas com alta suscetibilidade à cárie radicular, e o Diamino Fluoreto de Prata (>30%) para paralisar lesões ativas. Intervenções invasi-vas diretas são indicadas dependendo da situação clínica. Discussão: Nota-se uma falta de interesse em estudos primários sobre tratamentos para cárie radicular, criando assim uma lacuna em relação ao seu manejo, que reflete no nível de evidência detectado pelo consenso. Conclusão: Guias clíni-cos são importantes para reduzir a lacuna entre a pesquisa e a prática clínica. Essa tradução para o português facilitará o acesso dos dentistas bra-sileiros em relação a evidência consolidada até o momento para o manejo de cárie radicular.

Aim: Root caries are a problem in modern dentistry, but the lack of guidelines regarding their management is notable. We aim to develop and adapt a guide based on the translation of the recommendations of the international consensus as outlined by the European Organization for Caries Research (ORCA) and the European Federation of Conservative Dentistry (EFCD) for clinical decision-making in the intervention of the caries process in the elderly, with a focus on root caries. Materials and methods: The protocol for translating the recommendations of the international consensus consisted of the following steps: (1) initial translation, (2) synthesis of the translation, (3) back-translation, (4) review by an expert committee with cultural adaptation. Based on the translation, a guide was developed with guidelines for the treatment of root caries in Brazil. Results: To prevent new lesions, daily brushing with toothpaste >1,500ppm/F is recommended. Toothpaste with 5,000ppm/F or varnishes (>20,000ppm/F) may be recommended to paralyze active root lesions and for prevention in elderly people with high susceptibility to root caries, and Silver Diamine Fluoride (>30%) to paralyze active lesions. Direct invasive interventions are indicated depending on the clinical situation. Discussion: There is a lack of interest in primary studies on treatments for root caries, thus creating a gap in relation to its management, which is reflected by the level of evidence detected in the consensus. Conclusion: Clinical guidelines are important to reduce the gap between research and clinical practice. This translation into Portuguese will facilitate access by Brazilian dentists to the consolidated evidence gathered to date for the management of root caries.

The effect of coconut sugar on cariogenic traits in Streptococcus mutans / Efeito do Açúcar de Coco na cariogenicidade do Streptococcus mutans

Aim: The consumption of foods rich in sugar is linked to several non-communicable diseases, including dental caries. Coconut sugar has systemic benefits due to its lower glycemic indexes (GI) than other table sugars. However, there is currently no data regarding its ca-riogenic potential. This study aimed to evaluate the effect of coconut sugar on acidogenicity and adhesion of Streptococcus mutans in vitro, compared to sugarcane products. Materials and methods: Aliquots of cultu-res of S. mutans UA159 were resuspended in a buffer solution enriched with coconut sugar, crystal sugar (refined sugar), and minimally processed sugarcane (demerara light brown sugar and maskavo dark brown sugar), as well as positive (sucrose) and negative con-trols. The decrease in pH and its corresponding area under the curve (AUC; cm2) were evaluated for the analysis of acidogenicity. S.mutans was incubated in BHI supplemented with each sugar and the percentages of microbial adhesion were calculated. After testing data normality, the one-way ANOVA test (Bonferroni post hoc) was used to compare the AUC and the pro-portion of adhesion of each group. Results: Regarding the acidogenic potential, statistical differences were found only between the negative control versus all other groups (p<0.001). Likewise, no significant difference in adhesion was found between the tested sugars (p>0.05). Discussion: Although the tested sugars are marketed as "healthy products," their amount and frequency of usage should be controlled. Conclusion: Coconut su-gar presents a similar cariogenic potential to that of sugarcane products when acidogenicity and adhesion are evaluated. Coconut sugar is not indicated as a substitute for sucrose in the control of cariogenic activity.

Objetivo: O consumo de alimentos ricos em açúcar está associado a diversas doenças não transmissíveis, incluindo a doença cárie. O açúcar de coco tem benefícios sistêmi-cos devido aos seus índices glicêmicos (IG) mais baixos do que outros açúcares de mesa. No entanto, atualmente não há dados sobre seu potencial cariogênico. Esse estu-do teve como objetivo avaliar o efeito do açúcar do coco na acidogenicidade e adesão de Streptococcus mutans in vitro, em comparação com produtos derivados da cana--de-açúcar. Materiais e métodos: alíquotas de culturas de S. mutans UA159 foram suspensas em solução tampão enriquecida com açúcar de coco, açúcar cristal (açúcar refinado) e cana-de-açúcar minimamente processada (açúcar mascavo demerara claro e açúcar mascavo), além de controles positivo (sacarose) e negativo. A diminuição do pH e correspondente área sob a curva (AUC; cm2) fo-ram avaliadas na análise de acidogenicidade. S. mutansfoi incubado em BHI suplementado com cada tipo de açúcar e as porcentagens de adesão microbiana foram calculadas. Após testar a normalidade dos dados, o teste ANOVA de uma via (Bonferroni post hoc) foi utilizado para comparar a AUC e a proporção de adesão de cada grupo. Resultados: Em relação ao potencial acidogênico, diferenças estatísticas foram encontradas apenas entre o controle negativo versus todos os outros grupos (p <0,001). Não foi encontrada diferença significativa na adesão en-tre os açúcares testados (p> 0,05). Discussão: Apesar dos açúcares testados serem comercializados como "produtos saudáveis", sua quantidade e frequência de consumo deve ser controlada. Conclusão: O açúcar do coco apresenta potencial cariogênico semelhante à dos produtos da cana-de-açúcar quanto a acidogenicidade e a adesão. O açúcar de coco não é indicado como substituto da sacarose no controle da atividade cariogênica.

Streptococcus mutans e seu metabolismo a nível molecular no contexto ecológico da doença cárie / Streptococcus mutans and its metabolism at the molecular level in the ecological context of dental caries

Objetivo: Durante décadas, o Streptococcus mutans foi con-siderado o principal agente etiológico da doença cárie. Esta revisão apresentará seu histórico e metabolismo a nível molecular. Ao entender as vias metabólicas do S.mutans envolvidas no desenvolvimento de lesões cariosas, será possível desenvolver novos métodos de modulação de biofilmes no controle da doença cárie e elucidar a neces-sidade de continuar pesquisando essa bactéria. Revisão de literatura: Embora o S. mutans não constitua uma pro-porção significativa na colonização da microbiota bucal da dentição hígida, essa proporção aumenta quando há acidificação contínua do biofilme, associada ao excesso de carboidratos na dieta do hospedeiro. Isso ocorre devido a um conjunto de fatores de virulência, tais como, adesão, formação de biofilme, acidogenicidade, aciduricidade, atividades de proteases, produção de mutacinas e vias de transdução de sinal. Cada uma dessas propriedades, coordenadamente, alteram a ecologia do biofilme dental. Discussão: Ainda é relevante entender o metabolismo do S. mutans como microrganismo modelo em lesões cariosas devido a seus inúmeros fatores de virulência. Porém, no contexto da doença cárie como uma disbiose, estratégias terapêuticas antimicrobianas, mais especificamente anti-S.mutans, voltadas para a eliminação do microrganismo, po-dem não ser a chave do controle da doença cárie, enquanto a modulação do microbioma poderá se tornar o futuro das clínicas odontológicas. Conclusão: Biofilmes associados a doença cárie compreendem um ecossistema diverso, sugerindo uma etiologia polimicrobiana, porém, estudos futuros que visem à prospecção, ao desenvolvimento e à inter-relação do S. mutans com outros microrganismos e com o hospedeiro humano ainda são justificados a fim de desvendar a transição 'homeostase-disbiose'.

Aim: For decades, the Streptococcus mutans was consi-dered the main agent of caries. This review will show its history and metabolism at the molecular level. By understanding its metabolic pathways involved in the development of carious lesions, it can be possible to develop new methods of modulating biofilms in the control of caries, as well as to elucidate the need to continue researching this bacterium. Literature review: Although S. mutans does not constitute a significant proportion in the colonization of the oral microbiota of the sound dentition, its proportion increases when there is continuous acidification of the biofilm, asso-ciated with excess carbohydrates in the host diet. This is due to a set of virulence factors, such as adhesion, biofilm formation, acidogenicity, aciduricity, proteases activity, mutacins production and signal transduction pathways. Each of these properties coordinately alters the ecology of the dental biofilm. Discussion: It is still relevant to understand the metabolism of S. mutans as a model microorganism in carious lesions due to its numerous virulence factors. However, in the context of caries as a dysbiosis, antimicrobial therapeutic strategies, more specifically anti-S.mutans, aiming to eliminate the microorganism, may not be the key to caries control, and the microbiome modulation may become the future of dental clinics. Conclusion: Biofilms associated with caries disease comprise a diverse ecosystem, suggesting a polymicrobial etiolo-gy, however, future studies aimed at the prospection, development and interrelationship of S. mutans with other microorganisms and with the human host are still justified in order to unravel the 'homeos-tasis-dysbiosis' transition.

Minimum intervention oral care: defining the future of caries management

Abstract It is not uncommon that oral healthcare professionals and researchers interchange the use of the terms minimum/minimal intervention and minimally invasive dentistry. However, these terms apply to two different, but related, concepts. Minimum intervention dentistry, to be more appropriately addressed in this paper as minimum intervention oral care (MIOC), is an oral healthcare delivery framework that encompasses four interlinked clinical domains. These domains are: identifying disease - detection, longitudinal risk/susceptibility assessment, investigation, diagnosis and the development of a personalized care plan; prevention of lesions/control of disease - patient behaviour management, non-invasive remineralisation of the enamel and dentine, biofilm and diet control, micro-invasive sealants and infiltration techniques to arrest and reverse incipient lesions; minimally invasive operative procedures including selective carious dentine removal, the "5Rs" management of the tooth-restoration complex (review, refurbish, re-seal, repair and replace) amongst other restorative interventions; and tailored recall/review/re-assessment consultations. This framework includes that minimally invasive operative dentistry (MID), that although a critical operative clinical domain, should be viewed as one of the pillars of minimum intervention oral healthcare (MIOC), applied across all disciplines of restorative dentistry, not just caries management. The aim of this review is to clarify these differences and emphasize the importance of minimally invasive operative dentistry (MID) within the context of minimum intervention oral care (MIOC). MIOC is applicable to all disciplines within restorative dentistry, including clinical caries management.

Expression of inflammatory mediators in periodontitis and T2D patients: a systematic review and meta-analysis

Abstract The high concentration of glucose in the blood in Type 2 diabetes (T2D) may be related to either insulin resistance or insulin deficiency. Moreover, the literature points to periodontitis as the main oral disease caused by glycemia imbalance. The quantification of inflammatory markers in blood or saliva samples of T2D patients may represent a valuable tool in revealing how well an individual's immune system can respond to injuries and periodontal treatment. In addition, an evaluation of the cytokine expression is extremely relevant to help understand the connection between periodontitis and T2D. This systematic review and meta-analysis aimed to evaluate the expression of inflammatory markers in T2D patients with periodontitis, compared with non-diabetic patients with periodontitis. A total of 3,894 studies were retrieved after a systematic literature search, 15 of which were included in the systematic review, and 4 of these 15, in the meta-analysis. The results did not indicate any statistical difference between the groups regarding TNF-α and IL-6 markers. T2D patients with periodontitis had increased levels of IL-10, compared with non-diabetic individuals with periodontitis (p = 0.003). On the other hand, the IL-4 concentration in non-diabetic individuals with periodontitis was high, compared with the T2D group (p< 0.001). Several studies did not include quantitative results and were excluded from the meta-analysis. The high IL-10 expression and low IL-4 expression in the T2D group suggest an association between the level of these markers and the impairment of the immune response in T2D patients with periodontitis.

Prevalence and risk indicators for underlying dentin shadows among 12-year-old southern Brazilian schoolchildren

Abstract This study aimed to assess the prevalence, extent, and associated factors of underlying dentin shadows (UDS) in the occlusal surfaces of permanent posterior teeth among 12-year-old schoolchildren from southern Brazil. A population-based cross-sectional study that included a representative sample of 1,528 schoolchildren was conducted in Porto Alegre, southern Brazil (participation rate: 83.2%). A questionnaire on sociodemographic characteristics, behavioral habits, and access to dental services was sent to parents/legal guardians of each student selected. Caries examination included the recording of non-cavitated and cavitated lesions, including the presence of UDS, as defined by the ICDAS Group (i.e., a shadow of discolored dentin visible through an apparently intact enamel surface that may or may not show signs of localized breakdown - ICDAS Code 4). The association between predictor variables and UDS prevalence was assessed using survey Poisson regression models. The prevalence of UDS was 6.3% (95%CI = 1.2 -11.3). The mean number of affected teeth among the schoolchildren presenting UDS was 1.51 (95%CI = 1.40 -1.61), ranging from 1 to 6. Type of school (public, PR = 2.23, 95%CI = 1.22 -4.07) and caries experience (DMFT 1-2, PR = 2.41, 95%CI = 1.15 -5.04; DMFT ≥ 3, PR = 3.09, 95%CI = 1.52 -6.27) were significantly associated with UDS prevalence. In conclusion, this population-based cross-sectional study found a low prevalence of UDS in the occlusal surfaces of the permanent posterior teeth of 12-year-old southern Brazilian schoolchildren. Overall caries experience and type of school were associated with UDS in this population.

Bacterial analysis of biofilms from tooth root surfaces presenting different caries activity / Análise bacteriológica de biofilmes de superfícies dentárias radiculares apresentando diferentes estágios de atividade de cárie

This study evaluated the numbers and determined the proportion of mutans streptococci and Lactobacillus spp., which are possible relevant cariogenic organisms, in biofilms recovered from lesions at root surfaces with active caries lesions (ARC), inactive caries lesions, and sound root surfaces (SRS). Samples were cultured in MSB agar for mutans streptococci counts, Rogosa agar for Lactobacillus spp. counts, and brain-heart infusion agar for total viable anaerobic counts. After incubation, the number of colony-forming units (CFUs) was determined and compared between groups by the Mann-Whitney U test with a significance level set at 95%. The proportion of counts of mutans streptococci and Lactobacillus spp. in the total viable microorganisms was also analyzed by Chi-square test. Ninety samples (30 from each surface) from 37 patients were cultured and analyzed. The CFU was similar between mutans streptococci and Lactobacillus spp.These species were present in at least half of the samples and no difference was found in the frequency of isolation of these species. Only 6 samples showed a proportion of more than 10% of mutans streptococci; 4 of the samples were from ARC. Most (93%) SRS samples did not contain viable Lactobacillus spp. The data indicate the low counts of mutans streptococci and Lactobacillus spp. in root sur-faces, regardless of the activity of caries lesions.

O estudo analisou contagens e proporções de mutans strep-tococci e Lactobacillus spp., que podem ser microorganismos importantes em lesões de cárie radicular com diferentes atividades. Biofilmes foram coletados em três locais: ARC ­ superfície radicular com lesão ativa de cárie; IRC ­ superfícies radiculares com lesão inativa de cárie; SRS ­ superfícies de raizes hígidas. As amostras foram cultivadas em agar MSB para contagens de mutans streptococci; agar Rogosa para Lactobacillus spp., e agar BHI para contagens de microrganis-mos viáveis anaeróbicos totais. Após a incubação, o número de unidades formadoras de colônias (UFCs) foi determinado e comparado entre os grupos pelo teste de Mann-Whitney U test. O nível de significância foi estabelecido em 95%. A pro-porção de contagem de mutans streptococci e Lactobacillusspp. no total de microrganismos viáveis também foi analisado através do teste de qui-quadrado. Um total de 90 amostras de 37 pacientes foram cultivadas e analisadas: 30 amostras de ARC, 30 de IRC e 30 de SRS. Números de UFC foram seme-lhantes entre os grupos para ambos, mutans streptococci e Lactobacillus spp. Estas espécies estavam presentes em pelo menos metade de todas as amostras e nenhuma diferença foi encontrada na frequência de isolamento dessas espécies dentro dos grupos. Apenas 6 amostras apresentaram mais de 10% de mutans streptococci e 4 foram de ARC. Em relação aos Lactobacillus spp., 93% das amostras não apresentaram proporção dessas bactérias nas SRS. Mutans streptococci e Lactobacillus spp. estão presentes em baixa proporção nas superfícies radiculares, independentemente da atividade das lesões de cárie.

Análise da rugosidade superficial de uma resina composta tratada com gel fluoretado em diferentes pH / Surface roughness analysis of a composite treated with fluoridated gel at different pH

Objetivo: O flúor acidulado é utilizado no controle da doença cárie, mas seus efeitos na superfície de resinas compostas são controversos. O objetivo do estudo é avaliar a rugosidade superficial de uma resina composta após a aplicação tópica de flúor in vitro. Métodos: Espécimes de resina Vittra (FGM) foram preparados (8mmx1mm) e polidos. Após avaliação do pH de produtos fluoretados, o de maior e menor pH foram selecionados para os ex-perimentos. Foram testados três grupos experimentais (n = 15): grupo 1 ­ flúor gel acidulado a 1,23% (Nova DFL) (menor pH:4,48±0,32), grupo 2 ­ flúor gel neutro a 2% (Nova DFL) (maior pH:7,03±0,40), grupo 3 ­ sem exposi-ção ao flúor (controle negativo). O experimento simulou o protocolo padrão clínico de aplicação de flúor (uma aplicação por semana, durante um minuto, por quatro semanas). A rugosidade superficial foi mensurada antes e após o tratamento através de microscopia confocal e a característica superficial avaliada por imagens reconstru-ídas. Foi realizado teste ANOVA (post-hoc de Bonferroni) para comparar médias da rugosidade superficial entre grupos. Resultados: O grupo exposto ao flúor gel acidu-lado apresentou os maiores valores de rugosidade (Ra: 0,29±0,08), sendo maior que o grupo exposto ao gel neutro (Ra: 0,21±0,08) e controle (Ra: 0,23±0,05) (p < 0,05), que não apresentaram diferença estatística entre si. Conclusão: O grupo exposto ao flúor acidulado mostrou rugosidade superficial maior que o controle, porém essa diferença pode ser clinicamente irrelevante. Novos estudos são necessários para avaliar se tal alteração aumenta a adesão bacteriana e se a resina deve ser polida após aplicações tópicas de flúor.

Aim: Acidulated fluoride is widely used to control dental caries, however, its effects on composites surfaces are controversial. This study aimed to evaluate the surface roughness of a composite af-ter a fluoride topical application in vitro. Methods: Vittra (FGM) composite specimens were made (8mmx1mm) and polished. After pH evaluation of fluoridated products, the ones with the highest and lowest pH were selected for the experiments. Three experimental groups (n = 15) were tested: group 1 - 1.23% acidulated fluoride gel (Nova DFL) (pH: 4.48±0.32), group 2 - 2% neutral fluoride gel (Nova DFL) (pH: 7.03±0.40), group 3 - no fluoride exposure (negative control). The experiment simulated the standard clinical fluoride application protocol (one application of one minute per week,in four weeks). Surface roughness was measured before and after treatment by confocal microscopy and the surface characteristic evaluated by reconstructed images. ANOVA (Bonferroni post-hoc) test was performed to compare means of surface roughness between groups. Results: The group exposed to acidulated fluoride gel presented the highest values of roughness (Ra: 0.29±0.08), being larger than the group exposed to neutral fluoride gel (Ra: 0.21±0.08) and control (Ra: 0.23±0.05) (p < 0.05), which showed no statistical difference between them. Conclusion: It was concluded that the group exposed to acidula-ted fluoride showed greater surface roughness than the control group, but this difference appears to be clinically irrelevant. Further studies are needed to assess whether this change increases bacterial adhesion and whether the resin should be polished after topical fluoride applications.

Gene expression profile of Scardovia spp. in the metatranscriptome of root caries

Abstract A few investigations of caries biofilms have identified Scardovia spp.; however, little is known about its involvement in caries pathogenesis. The purpose of this study was to assess the gene expression profile of Scardovia spp. in root caries, and compare it with other microorganisms. Clinical samples from active root caries lesions were collected. Microbial mRNA was isolated and cDNA sequenced. The function and composition of the Scardovia were investigated using two methods: a) de novo assembly of the read data and mapping to contigs, and b) reads mapping to reference genomes. Pearson correlation was performed (p < 0.05). Proportion of Scardovia inopinata and Scardovia wiggsiae sequences ranged from 0-6% in the root caries metatranscriptome. There was a positive correlation between the transcriptome of Lactobacillus spp. and Scardovia spp. (r = 0.70; p = 0.03), as well as with other Bifidobacteriaceae (r = 0.91; p = 0.0006). Genes that code for fructose 6-phosphate phosphoketolase (the key enzyme for "Bifid shunt"), as well as ABC transporters and glycosyl-hydrolases were highly expressed. In conclusion, "Bifid shunt" and starch metabolism are involved in carbohydrate metabolism of S. inopinata and S. wiggsiae in root caries. There is a positive correlation between the metabolism abundance of Lactobacillus spp., Bifidobacteriaceae members, and Scardovia in root caries.

Are Brazilian dentists and dental students using the ICCC recommendations for caries management?

Abstract The aim of this study was to evaluate whether dentists and dental undergraduate students know the terminology of the International Caries Consensus Collaboration (ICCC), and make their restorative treatment decisions regarding carious tissue removal accordingly. Data collection was performed using an electronic questionnaire, considering: a) profile of the respondent; b) analysis of four clinical cases with respect to possible management strategies; and c) questions on cariology field terminology. Sample size consisted of 175 dentists and 66 last semester dental students. Statistical analyses were performed comparing profile, type of institution and dental specialty of the participants. Results showed students were less conservative and agreed less with the ICCC than dentists, and private schools, less than public institutions. Private institutions were 12% (95%CI = 0.833-0.949; p = 0.000) more likely to be less updated with the ICCC recommendations than public institutions, and dentists were 20% more likely to agree with them than students (95%CI = 1.118-1.302; p = 0.000). Dentists were 66% more likely to be conservative than students (95%CI = 0.203-0.554; p = 0.000); dentists and students who graduated or were graduating from public universities were twice as likely to be conservative as those from private universities (95%CI = 1.336-3.333; p = 0.001). In conclusion, students in the last semester are less conservative than dentists, and respondents who graduated or were graduating from public dental schools were more aligned with the current concepts of the ICCC. Several answers were not aligned with ICCC directives, thus showing that management of deep carious lesions still causes restorative therapeutic insecurity.

The role of Candida albicans in root caries biofilms: an RNA-seq analysis

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

Microbial load after selective and complete caries removal in permanent molars: a randomized clinical trial

Abstract The aim of this randomized clinical trial was to compare the remaining microbial load after treatments based on complete and selective caries removal and sealing. Patients with active carious lesions in a permanent molar were randomly allocated into 2 groups: a test group (selective caries removal-SCR; n=18) and a control group (complete caries removal - CCR; n=18). Dentin samples were collected following the excavation and three months after sealing. Streptococcus species, Streptococcus mutans, Lactobacillus species, and total viable microorganisms were cultured to count the viable cells and frequency of species isolation. CCR resulted in significant lower total viable microorganisms counts (p≤0.001), Streptococcus species (p≤0.001) and Lactobacillus species (p≤0.001) initially. However, after sealing, a decrease in total viable microorganisms, Streptococcus species, and Lactobacillus species in the SCR resulted in no difference between the groups after 3 months. In conclusion, selective caries removal is as effective as complete caries removal in reducing dentin bacterial load 3 months after sealing.

Resumo O objetivo deste ensaio clínico randomizado foi comparar os microrganismos remanescentes após tratamentos baseados em remoção total de tecido cariado e selamento e a remoção seletiva de tecido cariado e selamento. Pacientes com lesões de cárie ativas em molares permanentes foram divididos aleatoriamente em dois grupos: grupo teste (remoção seletiva de tecido cariado-SCR; n=18), e grupo de controle (remoção total de tecido cariado-CCR; n=18). Amostras de dentina foram obtidas após a remoção da tecido cariado e após 3 meses de selamento das cavidades. Streptococcus spp., Streptococcus mutans, Lactobacillus spp. e microrganismos viáveis totais foram cultivados para contagem de células e frequência de isolamento de espécies. CCR resultou em menores contagens totais de microorganismos viáveis (p≤0,001), Streptococcus spp. (p≤0,001) e Lactobacillus spp. (p≤0,001) inicialmente. Entretanto, após o selamento, uma redução significativa nas contagens totais de microrganismos viáveis, Streptococcus spp. e Lactobacillus spp. resultou em nenhuma diferença entre os grupos após 3 meses. Conclui-se que a remoção seletiva de cárie é tão seletiva quanto a remoção completa de cárie na redução da infecção dentinária após três meses com selamento da lesão.

Efeito da redução do aporte nutricional na diversidade genotípica e nos fatores de virulência de bactérias cariogênicas isoladas de dentina cariada selada / Stress starvation effect on the genotypic diversity and virulence factors of cariogenic bacteria isolated from sealed carious dentin

No tratamento de lesões profundas de cárie, a Remoção Parcial de Dentina Cariada (RPDC) e restauração tem sido proposta como alternativa conservadora para evitar perda de tecido dentário e exposição pulpar. Existe uma hipótese de que uma seleção de bactérias ocorre abaixo de restaurações devido a um acesso limitado de nutrientes. No entanto, há falta de conhecimento sobre a diversidade e potencial de virulência das bactérias cariogênicas residuais seladas abaixo de restaurações sobre dentina cariada. O objetivo deste estudo foi caracterizar Streptococcus mutans e lactobacilos isolados de dentina cariada antes e após o estresse nutricional por selamento da cavidade. S. mutans e lactobacilos foram obtidos por cultivo da dentina cariada de lesões cavitadas de quatro e seis pacientes, respectivamente. Duas amostras de dentina cariada foram coletadas e cultivadas por paciente: uma antes e outra após três meses de selamento da cavidade. Colônias de S. mutans e lactobacilos preditos foram selecionadas, isoladas e analisadas por coloração de Gram. Genes “housekeeping” foram utilizados na identificação da espécie (gtfB para S. mutans e pheS/rpoA/groEL/16SrRNA para lactobacilos) e a técnica de AP-PCR foi utilizada para genotipagem. A análise fenotípica (produção de ácido e de tolerância ao ácido) foi realizada. Um total de 48 isolados representativos de S. mutans foram analisados (31 antes e 17 após estresse nutricional por meio do selamento). O número de genótipos diferentes de S. mutans encontrado foi de nove e seis antes e após selamento, respectivamente. Pelo menos um dos genótipos encontrados antes do selamento foi também encontrado na dentina após o estresse nutricional por meio do selamento...

A hypothesis exists that a selection of bacteria occurs underneath restoration due to a limited access of nutrients. However, there is a lack of evidence regarding their role in the progression of carious process beneath restorations after Partial Dentin Caries Removal. It still unclear if the diversity and the virulence potential of the sealed bacteria remain the same after sealing. The aim of this study was to characterize Streptococcus mutans and Lactobacillus species isolated from caries dentin before and after starvation stress by cavity sealing. S. mutans and lactobacilli were obtained by culture of carious dentin from four and six patients, respectively. Two carious dentin samples were collected and cultured per patient: 1st before and 2nd after three months of cavity sealing. Presumptive S. mutans and lactobacilli were selected, isolated and analyzed by Gram staining. Housekeeping genes sequencing were used to the species identification (gtfB for S. mutans and pheS/rpoA/groEL/16SrRNA for lactobacilli) and Arbitrary Primer-PCR (AP-PCR) was used for genotyping. Phenotypic analysis (acid production and acid tolerance) was performed. A total of 48 representative S. mutans isolates were genotyped (31 before and 17 after the sealing). The number of different genotypes identified was nine and six before and after sealing, respectively. At least one of the genotypes found before the sealing was also found on dentin after the sealing in all patients. Regarding lactobacilli, it was analyzed 86 strains, 41 before and 45 after starvation stress by sealing. L. paracasei and L. rhamnosus prevailed and only four isolates did not belong to these species. A total of 27 and 15 different genotypes were found before and after sealing, respectively...